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Ce [37] on a C57Bl/6 background were bred at Massachusetts General

Ce [37] on a C57Bl/6 background were bred at Massachusetts General Hospital and housed in a specific pathogen-free microisolator environment. C57Bl/6 mice were obtained from the Jackson Laboratory. All experiments were performed according to 22948146 protocols approved by the Massachusetts General Hospital Subcommittee on Research Animal Care (OLAW Number: A3596-01).RNA Isolation and qPCRTotal RNA was isolated from mouse skin, and quantitative PCR was performed as described [38] with the Mx4000 Multiplex Quantitative PCR System (Stratagene). Primer sequences used for qPCR of b2m and CCL7 [39]; IL-4, IL-5, IL-13, IFN-c and IL-25 [40]; IL-17A, IL-17F, and IL-22 [41] have been published. The following additional primer pairs were used: CCL2 F-TGG CTC AGC CAG ATG CAG T and R-TTG GGA 23977191 TCA TCT TGC TGG TG; CCL12 F-GCT GGA CCA GAT GCG GTG and RCCG GAC GTG AAT CTT CTG CT; TSLP F-ACG GAT GGG GCT AAC TTA CAA and R-AGT CCT CGA TTT GCT CGA ACT.Intradermal IL-23 Injections and Ear Swelling MeasurementCutaneous inflammation was induced by injecting the ears of anesthetized mice every other day with 20 mL PBS alone or containing 500 ng IL-23 (R D Systems) using a 30-gauge needle attached to a 50 mL Hamilton syringe every other day for ten days. Ear swelling was measured each day immediately before injection, starting on day 0. Ear measurements were made using a pocket thickness gauge (Mitutoyo USA, Aurora, IL).Histology, Measurement of Epidermal Thickness and Eosinophil, Neutrophil and Mast Cell QuantitationFor histological assessment, ears were isolated and placed in 10 formalin. Formalin preserved ear skin was embedded in paraffin and sections were stained with H E. Images were acquired and an investigator blinded to the genotype of the animals determined the percent of eosinophils or neutrophilsTSLP Tissue Lysate ELISAEar skin was transferred into T-PER tissue protein extraction reagent (Thermo Scientific) containing a protease inhibitor cocktail (Roche) and homogenized using a Polytron (Kinematica:AG). Protein concentration was quantified using a BCA protein quantification assay. ELISA for TSLP protein was performed according to the manufacturer’s instructions (R D Systems).IL-23 Induces Th2 Inflammation in CCR22/2 MiceStatisticsComparisons were analyzed for statistical significance by Student’s t-test with Microsoft Excel software, with P,0.05 being considered significant.Results IL-23-induced Cutaneous Inflammation is More Severe in CCR22/2 Mice than in WT MiceExpression of the CCR2 Biotin N-hydroxysuccinimide ester manufacturer ligand, CCL2 by basal keratinocytes within psoriatic plaques has been detected [26,27], suggesting a role for CCR2 in psoriasis pathogenesis. To determine the requirement of CCR2 for development of psoriasis, we 114311-32-9 cost examined whether CCR22/2 mice are protected from the development of IL-23-induced psoriatic inflammation. We injected WT and CCR22/2 mice intradermally in the ear with IL-23 every other day. As has been reported previously, WT mice develop severe ear swelling following intradermal IL-23 injection [7,8,9]. Ear thickness increased by more than 150 mm compared with PBSinjected WT control mice twelve days after initiation of IL-23 injections (Figure 1). Contrary to our hypothesis, CCR22/2 mice actually developed more severe ear swelling than WT mice. Average ear thickness of CCR22/2 mice increased more than 300 mm on day 12 (Figure 1).pared to WT mice (Figure 2a, 3a). Additionally, whereas WT mice developed parakeratosis, the stratum corneum of CCR22/2 mice lacked nuclei (.Ce [37] on a C57Bl/6 background were bred at Massachusetts General Hospital and housed in a specific pathogen-free microisolator environment. C57Bl/6 mice were obtained from the Jackson Laboratory. All experiments were performed according to 22948146 protocols approved by the Massachusetts General Hospital Subcommittee on Research Animal Care (OLAW Number: A3596-01).RNA Isolation and qPCRTotal RNA was isolated from mouse skin, and quantitative PCR was performed as described [38] with the Mx4000 Multiplex Quantitative PCR System (Stratagene). Primer sequences used for qPCR of b2m and CCL7 [39]; IL-4, IL-5, IL-13, IFN-c and IL-25 [40]; IL-17A, IL-17F, and IL-22 [41] have been published. The following additional primer pairs were used: CCL2 F-TGG CTC AGC CAG ATG CAG T and R-TTG GGA 23977191 TCA TCT TGC TGG TG; CCL12 F-GCT GGA CCA GAT GCG GTG and RCCG GAC GTG AAT CTT CTG CT; TSLP F-ACG GAT GGG GCT AAC TTA CAA and R-AGT CCT CGA TTT GCT CGA ACT.Intradermal IL-23 Injections and Ear Swelling MeasurementCutaneous inflammation was induced by injecting the ears of anesthetized mice every other day with 20 mL PBS alone or containing 500 ng IL-23 (R D Systems) using a 30-gauge needle attached to a 50 mL Hamilton syringe every other day for ten days. Ear swelling was measured each day immediately before injection, starting on day 0. Ear measurements were made using a pocket thickness gauge (Mitutoyo USA, Aurora, IL).Histology, Measurement of Epidermal Thickness and Eosinophil, Neutrophil and Mast Cell QuantitationFor histological assessment, ears were isolated and placed in 10 formalin. Formalin preserved ear skin was embedded in paraffin and sections were stained with H E. Images were acquired and an investigator blinded to the genotype of the animals determined the percent of eosinophils or neutrophilsTSLP Tissue Lysate ELISAEar skin was transferred into T-PER tissue protein extraction reagent (Thermo Scientific) containing a protease inhibitor cocktail (Roche) and homogenized using a Polytron (Kinematica:AG). Protein concentration was quantified using a BCA protein quantification assay. ELISA for TSLP protein was performed according to the manufacturer’s instructions (R D Systems).IL-23 Induces Th2 Inflammation in CCR22/2 MiceStatisticsComparisons were analyzed for statistical significance by Student’s t-test with Microsoft Excel software, with P,0.05 being considered significant.Results IL-23-induced Cutaneous Inflammation is More Severe in CCR22/2 Mice than in WT MiceExpression of the CCR2 ligand, CCL2 by basal keratinocytes within psoriatic plaques has been detected [26,27], suggesting a role for CCR2 in psoriasis pathogenesis. To determine the requirement of CCR2 for development of psoriasis, we examined whether CCR22/2 mice are protected from the development of IL-23-induced psoriatic inflammation. We injected WT and CCR22/2 mice intradermally in the ear with IL-23 every other day. As has been reported previously, WT mice develop severe ear swelling following intradermal IL-23 injection [7,8,9]. Ear thickness increased by more than 150 mm compared with PBSinjected WT control mice twelve days after initiation of IL-23 injections (Figure 1). Contrary to our hypothesis, CCR22/2 mice actually developed more severe ear swelling than WT mice. Average ear thickness of CCR22/2 mice increased more than 300 mm on day 12 (Figure 1).pared to WT mice (Figure 2a, 3a). Additionally, whereas WT mice developed parakeratosis, the stratum corneum of CCR22/2 mice lacked nuclei (.